There are currently no treatments that specifically target bacterial biofilms, an inherently antibiotic-resistant phenotype which contributes to significant morbidity in chronic wound and device infections. However, solutions to this problem have likely evolved in nature, through antagonistic bacterial interspecies interactions on surfaces. This proposal aims to identify co-evolved molecules that provide defense against infection in a natural system in order to test our central hypothesis that specific strategies have evolved for this purpose while exerting less selection pressure for resistance. Marine sponges provide an excellent system to test this hypothesis. They are simple filter-feeding invertebrates that lack cellular immune systems, but they do possess a complex stable community of symbiotic bacteria talented in the biosynthesis of bioactive small molecules. Preliminary data collected by the applicant suggests that anti-biofilm compounds are induced in response to perturbations of the sponge microbiota. This project will integrate culture-independent meta-omics techniques to identify anti-biofilm agents made by sponge symbionts and determine their propensity to induce resistance. In the R21 phase, small molecules used to modulate bacterial communities in sponges will be identified, with the following specific aims: 1) Evaluate the dynamics of bacterial populations and small molecule expression in sponges; and 2) Evaluate the effect of induced small molecules on biofilms. Under the first aim, unbiased direct sequencing of environmental DNA (metagenomics) and metatranscriptomics will be used to reconstruct the individual genomes of multiple symbiotic bacteria and assess the differential expression of small molecule biosynthetic pathways in response to microbiome perturbations. The feasibility of this approach was established through pilot studies in another marine invertebrate. Under the second aim, induced compounds will be characterized, assayed for anti-biofilm activity and associated with biosynthetic pathways identified in Aim1. In the R33 phase, anti-biofilm agents identified in the R21 phase will be further developed with the following specific aims: 3) Establish a renewable supply for compounds identified in the R21 phase; and 4) Assess mechanism of action and potential selection pressures induced by identified compounds. The third aim will leverage the extensive sequencing data collected in the R21 phase to inform targeted isolation of small molecule producing strains or to express biosynthetic pathways heterologously. The fourth aim will use chemical genetics and continuous biofilm reactors to investigate compounds' mechanism of action and the evolutionary processes that lead to resistance. The outcomes of this proposal will be (i) a template for probing in situ chemical ecology interactions for drug discovery, and (ii) the discovery of non-bactericidal compounds that are active against biofilms.